DNA topoisomerases are the enzymes responsible for maintaining and controlling the topological state of DNA in the cell. They play an important role in replication, transcription and genetic recombination. Despite their importance, their structure and detailed mechanism of action are still unknown. This project is concerned with the study of the structure of Escherichia coli DNA topoisomerase I. The enzyme works by forming a transient phosphotyrosine covalent bond with DNA that allows for one DNA strand to pass through the other. In this way it changes the DNA linking number in steps of one. No external energy source is required as the bond energy is conserved. This project is relevant not only to the studies of topoisomerases but also to the studies of those proteins involved in re-arrangements of DNA and proteins that form covalent bonds to DNA. The long-term goals of this project are to study the structure and function of E. coli DNA topoisomerase I and its complexes with DNA. In particular, to study the active site atomic environment, the nature of the covalent bond with DNA, and the DNA-protein interactions. The objective of these studies is to obtain a detailed model for the catalytic mechanism of E. coli DNA topoisomerase I and the way it modifies the topology of DNA. To accomplish these goals the specific aims of this proposal are i) to study the structure of a 35 kD fragment of the enzyme containing the catalytic tyrosine and responsible for the cleavage of single stranded DNA. ii) To characterize the 35 kD fragment, study its DNA binding properties and sequence dependence of the cleavage reaction. We will study the formation of covalent complexes of the fragment with short oligonucleotides. iii) to study the structure of the same fragment complexed with short oligonucleotides. iv) To study the structure of the intact protein both with and without DNA. The structural work will be based mainly on x-ray crystallographic studies. Crystals of the 35 kD fragment have already been obtained and diffract to at least 2.8 A resolution. We will continue our studies of the single stranded DNA cleavage reaction by both the intact protein and the 35 kD fragment; we are particularly interested in obtaining covalent complexes with short oligonucleotides that are suitable for crystallographic work. The work on attempting to crystallize the intact protein will continue.